Study of growth factors and receptors in carcinoma

8/7/2019 Study of growth factors and receptors in carcinoma

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Study of growth factors and receptors in carcinoma

ex pleomorphic adenoma

Cristiane Furuse1, Lucyene Miguita1, Ana Cla udia Garcia Rosa1, Andresa Borges Soares1, Elizabeth FerreiraMartinez1, Albina Altemani2, Vera Cavalcanti de Arau jo1

1Department of Oral Pathology, Sao Leopoldo Mandic Institute and Research Center, Campinas, Sao Paulo, Brazil;2Department of

Pathology, School of Medicine, State University of Campinas (UNICAMP), Campinas, Sao Paulo, Brazil

SUMMARY

Carcinoma ex pleomorphic adenoma (CXPA) is a rare

malignant salivary gland tumor derived from a pre-

existing pleomorphic adenoma. It is a good model to

study the evolution of carcinogenesis, starting with in situ

areas to frankly invasive carcinoma. Growth factors are

associated with several biological and neoplastic pro-

cesses by transmembrane receptors. In order to investi-

gate, by immunohistochemistry, the expression of some

growth factors and its receptors [EGF receptor, fibro-

blast growth factor, fibroblast growth factor receptor 1,

fibroblast growth factor receptor 2, hepatocyte growth

factor, c-Met, transforming growth factor (TGF) b1,TGFbR-II and insulin-like growth factor receptor 1] in the

progression of CXPA, we have used ten cases of CXPA in

several degrees of invasion- intracapsular, minimally and

frankly invasive carcinoma- with only epithelial compo-

nent. Slides were qualitatively and semi-quantitatively

evaluated according to the percentage of stained tumor

cells from 0 to 3 (0 = less than 10%; 1 = 1025%; 2 = 25

50%; 3 = more than 50% of cells). Malignant epithelial

cells starting with in situ areas showed stronger expres-

sion than luminal cells of pleomorphic adenoma for all

antibodies. Most of the intracapsular, minimally and

frankly invasive CXPA presented score 3. However, score

2 was more evident in the frankly invasive one. In smallnests of invasive carcinoma, negative cells were observed

probably indicating that the proliferative process is re-

placed by the invasive mechanism. Altogether this data

infers that these factors may contribute to cell prolifer-

ation during initial phases of the tumor.

J Oral Pathol Med(2010)

Keywords: carcinoma ex pleomorphic adenoma; growth factors

Abbreviations: rRNA, ribosomal ribonucleic acid; ErbBHER,

epidermal growth factor receptor family; PDGFR-a, platelet

growth factor receptor type a; FGFR2b, fibroblast growth factor

receptor isoform 2b; FGFR2c, fibroblast growth factor receptor

isoform 2c; GTPase, guanosine triphosphate hydrolase enzyme;

GEP100, guanine-nucleotide exchange protein 100; TGF-a,

transforming growth factor a.

Introduction

According to the World Health Organization thecarcinoma ex pleomorphic adenoma (CXPA) is definedas an epithelium malignization derived from a pleomor-phic adenoma (1). The structural diversity of CXPAenables us to an important template study of theevolution of carcinogenesis, from benign to malignanttumor (27).

Several growth factors are involved in carcinomainitiation and progression including fibroblast growthfactor (FGF), hepatocyte growth factor (HGF), epider-mal growth factor (EGF), insulin-like growth factor(IGF) and transforming growth factor b (TGFb) (817).Growth factors and their receptors have been intensely

studied in tumorigenesis of mammary, skin, prostateand bladder carcinomas (1821), blood cancer (8, 22),neural astrocytoma (23), but rarely studied in salivarygland tumors (2427).

Among them, the FGF-2 is a member of a family ofheparan-biding polypeptides, localized in extracellularmatrix, cytoplasm and nucleus of the cells (2830). Itacts on cells by intracrine, paracrine and autocrinemechanisms (28, 31, 32) activating intracelullar path-ways by tyrosine kinase receptors (FGFR-1 and FGFR-2) (29, 30, 33, 34), or by internalization to cytoplasmand translocation to nucleus acting directly on nucleo-lus where FGF-2 participates in rRNA transcription(35, 36). In the nucleus, FGF-2 promotes mitogenic

Correspondence: Vera Cavalcanti de Araujo, Rua Vicente Leporace,1220 apt. 91, Sao Paulo, SP, Brazil, 04619-033. Tel: +55 11 5044 0762,Fax: +55 11 5041 2992, E-mail: [email protected] for publication September 30, 2009

doi: 10.1111/j.1600-0714.2009.00858.x

J Oral Pathol Med

2010 John Wiley & Sons A/S All rights reserved

interscience.wiley.com/journal/jop

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Oral Pathology & Medicine


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function, cell differentiation, angiogenesis, phenotypictransformation (35, 37), and survival of tumor and stemcells (8, 3840).

HGF is a cytokine that induces many biologicalfunctions not only in hepatocytes but also in manyepithelial cells (41, 42) by activating a tyrosine kinasesignaling cascade after binding to the proto-oncogenicC-Met receptor (43). In normal tissue, HGF plays a rolein the dynamic construction and reconstruction oftissues during organogenesis and tissue regeneration(44, 45). In mature tissues, HGF has an organotrophicrole in regeneration and protection of various tissues(44). HGF also has angiogenic activity for vascularendothelial cells (46). In tumor tissues, cells utilize thebiological actions of HGF for their dissociative, invasiveand metastatic behavior.

EGF receptor (EGFR) is a member of ErbBHERtransmembrane protein family that performs manybiological functions in normal cells, as proliferation,adhesion and migration (47, 48). Many growth factorsof EGF family can activate EGFR on cell surface,

resulting in a downstream cascade of proteins whichpromotespromoting signal transduction (48). In neo-plastic cells, the super-expression of receptor as well asmutations of its cytoplasmatic domain contributes toconstitutive signalization which appears to be relevant inthe growth and progression of many cancers (49).

IGF plays an important role in normal cellulargrowth and development and it has been implicatedin the regulation of tumor growth (5052). One ofits receptors, insulin-like growth factor receptor 1(IGFR-1), is a transmembrane receptor with tyrosinekinase activity, and regulates cell growth and meta-bolism (53, 54).

TGFb-1 family encompasses a group of structurally

related growth and differentiation factors which havediverse activities in the regulation of cell growth,differentiation, embryonic induction and morphogenesisin a wide range of cells and tissues (55, 56). It has alsobeen implicated in carcinogenesis progression in differ-ent tumors (5759). TGFb-1 signals through the type Ireceptor (TGFbR-I) and the type II receptor (TGFbR-II) (9, 12) and it usually stimulates mesenchymal cellsgrowth but inhibits epithelial cell proliferation (60). Incancer, it plays a major role by suppressing tumorgrowth in the early phase of neoplasia, while promotingtumor progression and metastasis in later phases (61).

Based on the important role of growth factors and

their receptors in many tumors, the aim of the presentstudy was to investigate the participation of thesegrowth factors in CXPA with different evolution stagesby immunohistochemistry.

Materials and methods

The present study protocol was approved by the EthicsCommittee of School of Medicine of the State Univer-sity of Campinas, SP, Brazil. The CXPA cases wereretrieved from the files of the Pathology Department atthe School of Medicine of the State University ofCampinas, Campinas, Brazil. The tumors were classified

according to the presence of epithelial andor myoepi-thelial cells using immunohistochemistry for cytokera-tins (epithelial cells), vimentin and a-smooth muscleactin (myoepithelial cells) as previously described inAltemani et al. (2). Tumors with epithelial componentswere selected in a total of 10 cases (Table 1) andclassified according to Brandwein et al. (62) taking intoaccount the extension of invasion beyond the previouspleomorphic adenoma capsule as intracapsular (withoutinvasion), minimally invasive (1.5 mm of invasion) andfrankly invasive. In addition, luminal cells from residualpleomorphic adenoma present in the CXPA specimensand 4 cases of pleomorphic adenoma without malignanttransformation, retrieved from the files of the Depart-ment of Oral Pathology of Sao Leopoldo MandicInstitute and Research Center, Campinas, SP, Brazil,were also analysed.

Serial sections, 3 lm thick, were obtained fromparaffin-embedded samples and the dewaxed sectionswere processed for epitope desmasking. Endogenousperoxidase was blocked by incubation in 3% hydrogen

peroxide. After washing, the sections were incubatedwith primary polyclonal antibodies to FGF-2, FGFR-1,FGFR-2, HGF-A, C-Met, TGFb-1, EGFR and IGFR-1, and primary monoclonal antibody to TGFbR-II(Table 2). Proper positive controls were utilized for eachantibody. Omission of the primary antibody constitutedthe negative control. Signal detection was performedusing the DAKO EnVision Peroxidase procedure(DAKO, Carpinteria, CA, USA), followed by a dia-minobenzidine chromogen solution and counterstainingwith Mayers hematoxylin.

The labeled sections were qualitatively and semi-quantitatively evaluated by six independent examiners.The qualitative analysis was performed evaluating the

positive cells in different areas including in situ andperipheral areas of both intracapsular and minimallyinvasive tumors, large and small groups of cells of thefrankly invasive carcinoma. For semi-quantitative eval-uation, we followed the recommendation of the Mem-bers of Ad-Hoc Committee on ImmunohistochemistryStandardization (63). The scores for the expression ofeach protein were assigned according to the percentage

Table 1 Sex, age, localization, and degree of invasion of thecarcinoma ex pleomorphic adenoma

Caseno. Sex

Age(years)

Salivarygland

Degree ofinvasion

1 Male 58 Parotid Intracapsular2 Female 50 Parotid Intracapsular3 Female 37 Submandibular Intracapsular4 Female 51 Parotid Intracapsular5 Female 65 Parotid Minimally invasive6 Female 43 Parotid Minimally invasive7 Male 74 Parotid Minimally invasive8 Female 62 Submandibular Frankly invasive9 Male 66 Parotid Frankly invasive

10 a a Parotid Frankly invasive

aData not available.


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of stained tumor cells from 0 to 3 (0,


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A B

C D

E F

Figure 1 Immunohistochemical staining of growth factors and receptors in carcinoma ex pleomorphic adenoma. (A) Positive staining of TGF b inmalignant cells of CXPA. Note negative expression in residual pleomorphic adenoma (200). (B) expression of HGFA in in situ areas ofintracapsular type (400). (C, D) Large blocks of carcinoma cells in minimally invasive type of CXPA. In (C), staining for IGFR (200), and in (D)for FGFR2 (200). (E, F) Small nests of carcinoma cells in minimally invasive type of CXPA. In (E), negative carcinoma cells are evident usingFGF2 antibody (400). In (F), EGFR expression is shown (400).

Table 3 Semi-quantitative analysis of the growth factors and their receptors in residual pleomorphic adenoma of carcinoma ex pleomorphicadenomas and in pleomorphic adenoma without malignant transformation

Tumors FGF-2 FGFR-1 FGFR-2 TGF b-1 TGF bR-II IGFR EGFR HGF-A c-Met

PA luminal cells 1 1 1 (nuclei) 0 0 0 1 3 0CXPA intracapsular

#1 3 2 2 3 3 3 3 3 3

#2 3 3 3 3 3 3 3 3 3#3 3 3 3 3 3 3 3 3 3#4 3 3 0 3 3 3 0 3 3

CXPA minimally invasive#5 3 3 3 3 3 3 3 3 3#6 3 3 3 3 3 3 3 3 3#7 3 3 3 3 3 3 3 3 3

CXPA frankly invasive#8 3 3 2 3 3 2 3 3 3#9 2 3 0 3 2 3 3 3 3#10 3 3 2 3 2 3 3 2 3

PA, pleomorphic adenoma; CXPA, carcinoma ex-pleomorphic adenoma.Scores: 0 = less than 10% of tumor cells; 1 = staining of 1025% of tumor cells; 2 = staining of 2550% of cells; 3 = staining of more than 50%of cells.


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FGFR-2 was not present in pleomorphic adenoma butwas strongly expressed in CXPA cases. FGFR-2 isassociated with increased risk of breast cancer (14, 74,78, 79). Gene amplification or missense mutation ofFGFR2 occurs in gastric cancer, lung cancer, breastcancer, ovarian cancer, and endometrial cancer (80).This evidences raise the possibility that this receptor,FGFR-2, might be related with a malignant phenotypeof epithelial cells.

In the present study, TGFb-1 was observed in allcarcinoma cells, but negative in epithelial and myo-epithelial pleomorphic adenoma cells. TGFb has adual role in carcinogenesis, initially it acts as a tumorsuppressor and causes growth arrest of epithelial cellsand in the early stages of cancer, but in an establishedtumor, TGF-b exerts an effect which contributes for thesurvival, progression and metastasis of the tumor bypromoting epithelial-mesenchymal transition (EMT),angiogenesis and escape from immune surveillance(12). TGFb-1 is found in adenocarcinoma (12), carci-noma-associated fibroblast (81), but negative in pleo-

morphic adenoma (25). In human breast cancer cells,TGFb has been shown to induce breast tumorigenesisand metastatic progression (82).

HGF-A and c-Met were strongly expressed in allCXPA cases. HGF-A (also known as scatter factor) andits receptor (c-Met, a proto-oncogene also known asHGFR), play an important role in angiogenesis andtumor growth (83). HGF is expressed in many malig-nant tumor as osteosarcoma, rabdomyosarcoma (11),adenoid cystic carcinoma (84, 85) and breast cancer (86).While c-Met overexpression is evidenced in colorectalcarcinomas, hepatocarcinomas, gastrinomas, and carci-nomas of the pancreas, stomach, prostate, ovary andbreast (11), and also found in lung adenocarcinoma,

colorectal, ovarian, head and neck squamous cellcarcinoma, liver glioma, renal clear cell and papillarycancer, stomach cancer, melanoma (87), papillary car-cinoma of thyroid (88), sporadic papillary renal cancer,childhood hepatocellular carcinoma, osteosarcoma andrabdomyosarcoma (11). In pleomorphic adenoma,HGF-A was also present, probably playing a role incell differentiation as demonstrated by Niranjan et al.(89).

In the present study, IGFR-1 was observed in allcarcinoma cells. Expression of IGF and IGFR-1 hasbeen detected in various human malignancies, such asbrain (90), colorectal (17), breast (50) and oral (91)

carcinomas, suggesting that these molecules induce cellproliferation and inhibition of apoptosis in an autocrineor paracrine manner (50, 54, 92, 93). In addition, IGFR-1 has shown to be involved in malignant transformationand the frequent detection of IGFR-1 expression inhuman cancer has fired the efforts to develop IGFR-1targeted therapy (51, 94, 95).

Invasive tumors showed absence of the staining insome small nests and in isolated cells. Only EGFR waspresent in these nests. The absence of the antibodiesFGF-2, FGFR-1, FGFR-2, HGF-A, c-Met, TGFb-Iand TGFbR-I in these areas is in accordance with otherstudies which have pointed out that the cells lose their

proliferative capacity in order to invade (96, 97). It isbelieved that cancer cells cannot move and proliferatesimultaneously, a mechanism known as the go-or growhypothesis (96). When there is no cell motility, tumorcell exclusively proliferates, and when movement initi-ates it changes the fitness landscape on which the cellpopulation evolves (6, 7, 97).

Meanwhile, the expression of studied growth factorswere lost in the small nests of cells, this fact was lessevident for EGFR. The phosphorylation of EGFRactivates multiple biological processes including cellmotility and invasion, by EGFR-GEP100-Arf6 path-way, activating matrix invasion and deranging E-cadh-erin (98). Advanced stage colorectal carcinoma presentssignificantly higher co-expression of EGFR, TGF-a andprotein S6K, a ribosomal protein that regulates celladhesion and invasiveness (99). EGFR is vastly studiedin breast tumors (98, 100, 101), colorectal cancer (17),pancreatic and oral carcinoma (100), gastric carcinoma(102) and also salivary gland tumors (103105). Severaltherapies have been developed to inactivate the EGFR

pathway including monoclonal antibodies against theextracellular domain of EGFR (106).In conclusion, the obtained results demonstrated that

carcinoma cells from CXPA presented the expression ofall growth factors and receptors in a derisive way. Weobserved that EGFR, FGF-2, FGFR-1, FGFR-2,HGF-A, c-Met, TGFb-1, TGFbR-II and IGFR-1 weremore expressed in carcinomatous cells of CXPA than inepithelial cells of pleomorphic adenoma ductal areas.Moreover, this expression was more intense in intra-capsular and minimally invasive CXPA, than franklyinvasive ones, probably contributing to cell proliferationduring initial phases of the tumor and demonstrating theself-sufficiency of carcinomatous cells. In frankly inva-

sive tumors we also identified strong EGFR expressionin small nests of peripheral borders of invasive CXPA,indicating that this receptor may be related to invasive-ness and cell detachment of CXPA.

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Acknowledgements

The authors wish to thank Audrey Jordao Basso and Jeruza Pinheiro da

Silveira Bossonaro for their excellent technical expertise and assistance.

This work was supported by FAPESP grant no. 0407960-0

Conflict of interest statement

None declared.


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